1. Field of the Invention
This invention relates to the inhibition of histone deacetylase. More particularly, the invention relates to compounds and methods for inhibiting histone deacetylase enzymatic activity.
2. Summary of the Related Art
In eukaryotic cells, nuclear DNA associates with histones to form a compact complex called chromatin. The histones constitute a family of basic proteins which are generally highly conserved across eukaryotic species. The core histones, termed H2A, H2B, H3, and H4, associate to form a protein core. DNA winds around this protein core, with the basic amino acids of the histones interacting with the negatively charged phosphate groups of the DNA. Approximately 146 base pairs of DNA wrap around a histone core to make up a nucleosome particle, the repeating structural motif of chromatin.
Csordas, Biochem. J., 286: 23-38 (1990) teaches that histones are subject to posttranslational acetylation of the xcex5-amino groups of N-terminal lysine residues, a reaction that is catalyzed by histone acetyl transferase (HAT1). Acetylation neutralizes the positive charge of the lysine side chain, and is thought to impact chromatin structure. Indeed, Taunton et al., Science, 272: 408-411 (1996), teaches that access of transcription factors to chromatin templates is enhanced by histone hyperacetylation. Taunton et al. further teaches that an enrichment in underacetylated histone H4 has been found in transcriptionally silent regions of the genome.
Histone acetylation is a reversible modification, with deacetylation being catalyzed by a family of enzymes termed histone deacetylases (HDACs). Grozinger et al., Proc. Natl. Acad. Sci. USA, 96: 4868-4873 (1999), teaches that HDACs may be divided into two classes, the first represented by yeast Rpd3-like proteins, and the second represented by yeast Hda1-like proteins. Grozinger et al. also teaches that the human HDAC1, HDAC2, and HDAC3 proteins are members of the first class of HDACs, and discloses new proteins, named HDAC4, HDAC5, and HDAC6, which are members of the second class of HDACs. Kao et al., Genes and Dev., 14: 55-66 (2000), discloses HDAC7, a new member of the second class of HDACs. Van den Wyngaert, FEBS, 478: 77-83 (2000) discloses HDAC8, a new member of the first class of HDACs.
Richon et al., Proc. Natl. Acad. Sci. USA, 95: 3003-3007 (1998), discloses that HDAC activity is inhibited by trichostatin A (TSA), a natural product isolated from Streptomyces hygroscopicus, and by a synthetic compound, suberoylanilide hydroxamic acid (SAHA). Yoshida and Beppu, Exper. Cell Res., 177: 122-131 (1988), teaches that TSA causes arrest of rat fibroblasts at the G1 and G2 phases of the cell cycle, implicating HDAC in cell cycle regulation. Indeed, Finnin et al., Nature, 401: 188-193 (1999), teaches that TSA and SAHA inhibit cell growth, induce terminal differentiation, and prevent the formation of tumors in mice.
These findings suggest that inhibition of HDAC activity represents a novel approach for intervening in cell cycle regulation and that HDAC inhibitors have great therapeutic potential in the treatment of cell proliferative diseases or conditions. To date, only a few inhibitors of histone deacetylase are known in the art. There is thus a need to identify additional HDAC inhibitors and to identify the structural features required for potent HDAC inhibitory activity.
The invention provides compounds and methods for treating cell proliferative diseases. In particular, the invention provides new inhibitors of histone deacetylase enzymatic activity.
In a first aspect, therefore, the invention provides novel inhibitors of histone deacetylase. In one embodiment, the novel inhibitors of histone deacetylase are represented by formula (1):
Cyxe2x80x94L1xe2x80x94Arxe2x80x94Y1xe2x80x94C(O)xe2x80x94NHxe2x80x94Zxe2x80x83xe2x80x83(1)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted;
L1 is xe2x80x94(CH2)mxe2x80x94Wxe2x80x94, where m is 0, 1, 2, 3, or 4, and W is selected from the group consisting of xe2x80x94C(O)NHxe2x80x94, xe2x80x94S(O)2NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94NHS(O)2xe2x80x94, and xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted;
Y1 is a chemical bond or a straight- or branched-chain saturated alkylene, wherein said alkylene may be optionally substituted; and
Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation;
provided that when L1 is xe2x80x94C(O)NHxe2x80x94, Y1 is xe2x80x94(CH2)nxe2x80x94, n being 1, 2, or 3, and Z is xe2x80x94Oxe2x80x94M, then Cy is not aminophenyl, dimethylaminophenyl, or hydroxyphenyl; and further provided that when L1 is xe2x80x94C(O)NHxe2x80x94 and Z is pyridyl, then Cy is not substituted indolinyl.
In a second embodiment, the novel inhibitors of histone deacetylase are represented by formula (2):
Cyxe2x80x94L2xe2x80x94Arxe2x80x94Y2xe2x80x94C(O)NHxe2x80x94Zxe2x80x83xe2x80x83(2)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted, provided that Cy is not a (spirocycloalkyl)heterocyclyl;
L2 is C1-C6 saturated alkylene or C2-C6 alkenylene, wherein the alkylene or alkenylene optionally may be substituted, provided that L2 is not xe2x80x94C(O)xe2x80x94, and wherein one of the carbon atoms of the alkylene optionally may be replaced by a heteroatom moiety selected from the group consisting of O; NRxe2x80x2, Rxe2x80x2 being alkyl, acyl, or hydrogen; S; S(O); or S(O)2;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted; and
Y2 is a chemical bond or a straight- or branched-chain saturated alkylene, which may be optionally substituted, provided that the alkylene is not substituted with a substituent of the formula xe2x80x94C(O)R wherein R comprises an xcex1-amino acyl moiety; and
Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation;
provided that when the carbon atom to which Cy is attached is oxo substituted, then Cy and Z are not both pyridyl.
In a third embodiment, the novel inhibitors of histone deacetylase are represented by formula (3):
Cyxe2x80x94L3xe2x80x94Arxe2x80x94Y3xe2x80x94C(O)NHxe2x80x94Zxe2x80x83xe2x80x83(3)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted, provided that Cy is not a (spirocycloalkyl)heterocyclyl;
L3 is selected from the group consisting of
(a) xe2x80x94(CH2)mxe2x80x94Wxe2x80x94, where m is 0, 1, 2, 3, or 4, and W is selected from the group consisting of xe2x80x94C(O)NHxe2x80x94, xe2x80x94S(O)2NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94NHS(O)2xe2x80x94, and xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94; and
(b) C1-C6 alkylene or C2-C6 alkenylene, wherein the alkylene or alkenylene optionally may be substituted, provided that L3 is not xe2x80x94C(O)xe2x80x94, and wherein one of the carbon atoms of the alkylene optionally may be replaced by O; NRxe2x80x2, Rxe2x80x2 being alkyl, acyl, or hydrogen; S; S(O); or S(O)2;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted; and
Y3 is C2 alkenylene or C2 alkynylene, wherein one or both carbon atoms of the alkenylene optionally may be substituted with alkyl, aryl, alkaryl, or aralkyl; and
Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation;
provided that when Cy is unsubstituted phenyl, Ar is not phenyl wherein L3 and Y3 are oriented ortho or meta to each other.
In a fourth embodiment, the novel histone deacetylase inhibitor is selected from the group represented by formulae (4)-(6): 
In a second aspect, the invention provides a pharmaceutical composition comprising an inhibitor of histone deacetylase represented by any one of formulae (1)-(6) and a pharmaceutically acceptable carrier, excipient, or diluent.
In a third aspect, the invention provides methods for inhibiting histone deacetylase in a cell, comprising contacting a cell in which inhibition of histone deacetylase is desired with an inhibitor of histone deacetylase. In a first embodiment according to this aspect of the invention, the inhibitor of histone deacetylase is represented by formula (1):
Cyxe2x80x94L1xe2x80x94Arxe2x80x94Y1xe2x80x94C(O)xe2x80x94NHxe2x80x94Zxe2x80x83xe2x80x83(1)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted;
L1 is xe2x80x94(CH2)mxe2x80x94Wxe2x80x94, where m is 0, 1, 2, 3, or 4, and W is selected from the group consisting of xe2x80x94C(O)NHxe2x80x94, xe2x80x94S(O)2NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94NHS(O)2xe2x80x94, and xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted;
Y1 is a chemical bond or a straight- or branched-chain saturated alkylene, wherein said alkylene may be optionally substituted; and
Z is selected from the group consisting of anilinyl, pyridyl, 2-thioxo-1,3,4-thiadiazol-2-yl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation;
provided that when L1 is xe2x80x94C(O)NHxe2x80x94, Y is xe2x80x94(CH2)nxe2x80x94, n being 1, 2, or 3, and Z is xe2x80x94Oxe2x80x94M, then Cy is not aminophenyl, dimethylaminophenyl, or hydroxyphenyl.
In a second embodiment according to this aspect of the invention, the inhibitor of histone deacetylase is represented by formula (2)
Cyxe2x80x94L2xe2x80x94Arxe2x80x94Y2xe2x80x94C(O)NHxe2x80x94Zxe2x80x83xe2x80x83(2)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted;
L2 is C1-C6 saturated alkylene or C2-C6 alkenylene, either of which may be optionally substituted;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted; and
Y2 is a chemical bond or a straight- or branched-chain saturated alkylene, which may be optionally substituted, provided that the alkylene is not substituted with a substituent of the formula xe2x80x94C(O)R wherein R comprises an xcex1-amino acyl moiety; and
Z is selected from the group consisting of anilinyl, pyridyl, 2-thioxo-1,3,4-thiadiazol-2-yl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation.
In a third embodiment according to this aspect of the invention, the inhibitor of histone deacetylase is represented by formula (3):
xe2x80x83Cyxe2x80x94L3xe2x80x94Arxe2x80x94Y3xe2x80x94C(O)NHxe2x80x94Zxe2x80x83xe2x80x83(3)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted, provided that Cy is not a (spirocycloalkyl)heterocyclyl;
L3 is selected from the group consisting of
(a) xe2x80x94(CH2)mxe2x80x94Wxe2x80x94, where m is 0, 1, 2, 3, or 4, and W is selected from the group consisting of xe2x80x94C(O)NHxe2x80x94, xe2x80x94S(O)2NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94NHS(O)2xe2x80x94, and xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94; and
(b) C1-C6 alkylene or C2-C6 alkenylene, wherein the alkylene or alkenylene optionally may be substituted, provided that L3 is not xe2x80x94C(O)xe2x80x94, and wherein one of the carbon atoms of the alkylene optionally may be replaced by O; NRxe2x80x2, Rxe2x80x2 being alkyl, acyl, or hydrogen; S; S(O); or S(O)2;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted; and
Y3 is C2 alkenylene or C2 alkynylene, wherein one or both carbon atoms of the alkenylene optionally may be substituted with alkyl, aryl, alkaryl, or aralkyl; and
Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation;
provided that when Cy is unsubstituted phenyl, Ar is not phenyl wherein L3 and Y3 are oriented ortho or meta to each other.
In a fourth embodiment according to this aspect of the invention, the novel histone deacetylase inhibitor is selected from the group represented by formulae (4)-(6): 
The invention provides compounds and methods for inhibiting histone deacetylase enzymatic activity. The invention also provides compositions and methods for treating cell proliferative diseases and conditions. The patent and scientific literature referred to herein establishes knowledge that is available to those with skill in the art. The issued patents, applications, and references that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of inconsistencies, the present disclosure will prevail.
For purposes of the present invention, the following definitions will be used:
As used herein, the terms xe2x80x9chistone deacetylasexe2x80x9d and xe2x80x9cHDACxe2x80x9d are intended to refer to any one of a family of enzymes that remove acetyl groups from the xcex5-amino groups of lysine residues at the N-terminus of a histone. Unless otherwise indicated by context, the term xe2x80x9chistonexe2x80x9d is meant to refer to any histone protein, including H1, H2A, H2B, H3, H4, and H5, from any species. Preferred histone deacetylases include class I and class II enzymes. Preferably the histone deacetylase is a human HDAC, including, but not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, and HDAC-8. In some other preferred embodiments, the histone deacetylase is derived from a protozoal or fungal source.
The term xe2x80x9chistone deacetylase inhibitorxe2x80x9d or xe2x80x9cinhibitor of histone deacetylasexe2x80x9d is used to identify a compound having a structure as defined herein, which is capable of interacting with a histone deacetylase and inhibiting its enzymatic activity. Inhibiting histone deacetylase enzymatic activity means reducing the ability of a histone deacetylase to remove an acetyl group from a histone. In some preferred embodiments, such reduction of histone deacetylase activity is at least about 50%, more preferably at least about 75%, and still more preferably at least about 90%. In other preferred embodiments, histone deacetylase activity is reduced by at least 95% and more preferably by at least 99%.
Preferably, such inhibition is specific, i.e., the histone deacetylase inhibitor reduces the ability of a histone deacetylase to remove an acetyl group from a histone at a concentration that is lower than the concentration of the inhibitor that is required to produce another, unrelated biological effect. Preferably, the concentration of the inhibitor required for histone deacetylase inhibitory activity is at least 2-fold lower, more preferably at least 5-fold lower, even more preferably at least 10-fold lower, and most preferably at least 20-fold lower than the concentration required to produce an unrelated biological effect.
The term xe2x80x9calkylxe2x80x9d as employed herein refers to straight and branched chain aliphatic groups having from 1 to 12 carbon atoms, preferably 1-8 carbon atoms, and more preferably 1-6 carbon atoms, which may be optionally substituted with one, two or three substituents. Unless otherwise apparent from context, the term xe2x80x9calkylxe2x80x9d is meant to include saturated, unsaturated, and partially unsaturated aliphatic groups. When unsaturated groups are particularly intended, the terms xe2x80x9calkenylxe2x80x9d or xe2x80x9calkynylxe2x80x9d will be used. When only saturated groups are intended, the term xe2x80x9csaturated alkylxe2x80x9d will be used. Preferred saturated alkyl groups include, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.
An xe2x80x9calkylenexe2x80x9d group is an alkyl group, as defined hereinabove, that is positioned between and serves to connect two other chemical groups. Preferred alkylene groups include, without limitation, methylene, ethylene, propylene, and butylene.
The term xe2x80x9ccycloalkylxe2x80x9d as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
An xe2x80x9carylxe2x80x9d group is a C6-C14 aromatic moiety comprising one to three aromatic rings, which may be optionally substituted. Preferably, the aryl group is a C6-C10 aryl group. Preferred aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl. An xe2x80x9caralkylxe2x80x9d or xe2x80x9carylalkylxe2x80x9d group comprises an aryl group covalently linked to an alkyl group, either of which may independently be optionally substituted or unsubstituted. Preferably, the aralkyl group is (C1-C6)alk(C6-C10)aryl, including, without limitation, benzyl, phenethyl, and naphthylmethyl. An xe2x80x9calkarylxe2x80x9d or xe2x80x9calkylarylxe2x80x9d group is an aryl group having one or more alkyl substituents. Examples of alkaryl groups include, without limitation, tolyl, xylyl, mesityl, ethylphenyl, tert-butylphenyl, and methylnaphthyl.
An xe2x80x9carylenexe2x80x9d group is an aryl group, as defined hereinabove, that is positioned between and serves to connect two other chemical groups. Preferred arylene groups include, without limitation, phenylene and naphthylene. The term xe2x80x9carylenexe2x80x9d is also meant to include heteroaryl bridging groups, including, but not limited to, benzothienyl, benzofuryl, quinolyl, isoquinolyl, and indolyl.
A xe2x80x9cheterocyclylxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d group is a ring structure having from about 3 to about 8 atoms, wherein one or more atoms are selected from the group consisting of N, O, and S. The heterocyclic group may be optionally substituted on carbon at one or more positions. The heterocyclic group may also independently be substituted on nitrogen with alkyl, aryl, aralkyl, alkylcarbonyl, alkylsulfonyl, arylcarbonyl, arylsulfonyl, alkoxycarbonyl, aralkoxycarbonyl, or on sulfur with oxo or lower alkyl. Preferred heterocyclic groups include, without limitation, epoxy, aziridinyl, tetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl, thiazolidinyl, oxazolidinyl, oxazolidinonyl, and morpholino. In certain preferred embodiments, the heterocyclic group is fused to an aryl or heteroaryl group. Examples of such fused heterocyles include, without limitation, tetrahydroquinoline and dihydrobenzofuran.
As used herein, the term xe2x80x9cheteroarylxe2x80x9d refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14xcfx80 electrons shared in a cyclic array; and having, in addition to carbon atoms, between one and about three heteroatoms selected from the group consisting of N, O, and S. Preferred heteroaryl groups include, without limitation, thienyl, benzothienyl, furyl, benzofuryl, dibenzofuryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, quinoxalinyl, tetrazolyl, oxazolyl, thiazolyl, and isoxazolyl.
As employed herein, a xe2x80x9csubstitutedxe2x80x9d alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is one having between one and about four, preferably between one and about three, more preferably one or two, non-hydrogen substituents. Suitable substituents include, without limitation, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as employed herein refers to chlorine, bromine, fluorine, or iodine.
As herein employed, the term xe2x80x9cacylxe2x80x9d refers to an alkylcarbonyl or arylcarbonyl substituent.
The term xe2x80x9cacylaminoxe2x80x9d refers to an amide group attached at the nitrogen atom. The term xe2x80x9ccarbamoylxe2x80x9d refers to an amide group attached at the carbonyl carbon atom. The nitrogen atom of an acylamino or carbamoyl substituent may be additionally substituted. The term xe2x80x9csulfonamidoxe2x80x9d refers to a sulfonamide substituent attached by either the sulfur or the nitrogen atom. The term xe2x80x9caminoxe2x80x9d is meant to include NH2, alkylamino, arylamino, and cyclic amino groups.
The term xe2x80x9cureidoxe2x80x9d as employed herein refers to a substituted or unsubstituted urea moiety.
In a first aspect, the invention provides novel inhibitors of histone deacetylase. In a first embodiment, the novel inhibitors of histone deacetylase are represented by formula (1):
Cyxe2x80x94L1xe2x80x94Arxe2x80x94Y xe2x80x94C(O)xe2x80x94NHxe2x80x94Zxe2x80x83xe2x80x83(1)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted;
L1 is xe2x80x94(CH2)mxe2x80x94Wxe2x80x94, where m is 0, 1, 2, 3, or 4, and W is selected from the group consisting of xe2x80x94C(O)NHxe2x80x94, xe2x80x94S(O)2NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94NHS(O)2xe2x80x94, and xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted;
Y1 is a chemical bond or a straight- or branched-chain saturated alkylene, wherein said alkylene may be optionally substituted; and
Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation;
provided that when L1 is xe2x80x94C(O)NHxe2x80x94, Y is xe2x80x94(CH2)nxe2x80x94, n being 1, 2, or 3, and Z is xe2x80x94Oxe2x80x94M, then Cy is not aminophenyl, dimethylaminophenyl, or hydroxyphenyl; and further provided that when L1 is xe2x80x94C(O)NHxe2x80x94 and Z is pyridyl, then Cy is not substituted indolinyl.
In certain preferred embodiments, Cy is C6-C14 aryl, more preferably C6-C10 aryl, and most preferably phenyl or naphthyl, any of which may be optionally substituted. In certain other preferred embodiments, Cy is heteroaryl. In some preferred embodiments, the heteroaryl group is selected from the group consisting of thienyl, benzothienyl, furyl, benzofuryl, quinolyl, isoquinolyl, and thiazolyl, any of which may be optionally substituted. In certain particularly preferred embodiments, Cy is selected from the group consisting of phenyl, naphthyl, thienyl, benzothienyl, and quinolyl, any of which may be optionally substituted.
L1 is xe2x80x94(CH2)mxe2x80x94Wxe2x80x94, where m is 0, 1, 2, 3, or 4, and W is selected from the group consisting of xe2x80x94C(O)NHxe2x80x94, xe2x80x94S(O)2NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94NHS(O)2xe2x80x94, and xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94. Preferably, m is 0, 1, or 2, more preferably 0 or 1.
Preferably, Ar is C6-C14 arylene, more preferably C6-C10 arylene, any of which may be additionally substituted. In certain preferred embodiments, Ar is phenylene, preferably 4-phenylene. In some preferred embodiments, the phenylene is fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which groups also may be optionally substituted.
Y1 is a chemical bond or is a straight- or branched-chain alkylene, which may be optionally substituted. In some preferred embodiments, Y1 is a chemical bond, and the group xe2x80x94C(O)NHxe2x80x94Z is directly attached to Ar. In some other preferred embodiments, Y1 is alkylene, preferably saturated alkylene. Preferably, the saturated alkylene is C1-C8 alkylene, more preferably C1-C6 alkylene, still more preferably C1-C3 alkylene, and yet still more preferably C1-C2 alkylene, any of which may be optionally substituted. In some particularly preferred embodiments, Y1 is methylene.
Substituted alkyl, aryl, heterocyclyl, or heteroaryl groups have one or more, preferably between one and about three, more preferably one or two substituents, which are preferably selected from the group consisting of C1-C6 alkyl, preferably C1-C4 alkyl; halo, preferably Cl, Br, or F; haloalkyl, preferably (halo)1-5(C1-C6)alkyl, more preferably (halo)1-5(C1-C3)alkyl, and most preferably CF3; C1-C6 alkoxy, preferably methoxy, ethoxy, or benzyloxy; C6-C10 aryloxy, preferably phenoxy; C1-C6 alkoxycarbonyl, preferably C1-C3 alkoxycarbonyl, most preferably carbomethoxy or carboethoxy; C6-C10 aryl, preferably phenyl; (C6-C10)ar(C1-C6)alkyl, preferably (C6-C10)ar(C1-C3)alkyl, more preferably benzyl, naphthylmethyl or phenethyl; hydroxy(C1-C6)alkyl, preferably hydroxy(C1-C3)alkyl, more preferably hydroxymethyl; amino(C1-C6)alkyl, preferably amino(C1-C3)alkyl, more preferably aminomethyl; (C1-C6)alkylamino, preferably methylamino, ethylamino, or propylamino; di-(C1-C6)alkylamino, preferably dimethylamino or diethylamino; (C1-C6)alkylcarbamoyl, preferably methylcarbamoyl, dimethylcarbamoyl, or benzylcarbamoyl; (C6-C10)arylcarbamoyl, preferably phenylcarbamoyl; (C1-C6)alkaneacylamino, preferably acetylamino; (C6-C10)areneacylamino, preferably benzoylamino; (C1-C6)alkanesulfonyl, preferably methanesulfonyl; (C1-C6)alkanesulfonamido, preferably methanesulfonamido; (C6-C10)arenesulfonyl, preferably benzenesulfonyl or toluenesulfonyl; (C6-C10)arenesulfonamido, preferably benzenesulfonyl or toluenesulfonyl; (C6-C10)ar(C1-C6)alkylsulfonamido, preferably benzylsulfonamido; C1-C6 alkylcarbonyl, preferably C1-C3 alkylcarbonyl, more preferably acetyl; (C1-C6)acyloxy, preferably acetoxy; cyano; amino; carboxy; hydroxy; ureido; and nitro. One or more carbon atoms of an alkyl, cycloalkyl, or heterocyclyl group may also be optionally substituted with an oxo group.
In some particularly preferred embodiments, Cy is a phenyl, naphthyl, thienyl, benzothienyl, or quinolyl moiety which is unsubstituted or is substituted by one or two substituents independently selected from the group consisting of C1-C4 alkyl, C1-C4 haloalkyl, C6-C10 aryl, (C6-C10)ar(C1-C6)alkyl, halo, nitro, hydroxy, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, carboxy, and amino.
In some preferred embodiments,Z is anilinyl or pyridyl, preferably 2-anilinyl or 2-pyridyl. In some other preferred embodiments, Z is thiadiazolyl, preferably 1,3,4-thiadiazol-2-yl, and more preferably a 5-substituted-1,3,4-thiadiazol2-yl. The thiadiazolyl is preferably substituted with a substituent selected from the group consisting of thiol, trifluoromethyl, amino, and sulfonamido.
In still other preferred embodiments, Z is xe2x80x94Oxe2x80x94M, wherein M is hydrogen or any pharmaceutically acceptable cation. Examples of pharmaceutically acceptable cations include, without limitation, sodium, potassium, magnesium, and calcium.
In a second embodiment, the invention provides novel inhibitors of histone deacetylase represented by formula (2):
Cyxe2x80x94L2xe2x80x94Arxe2x80x94Y2xe2x80x94C(O)NHxe2x80x94Zxe2x80x83xe2x80x83(2)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted, provided that Cy is not a (spirocycloalkyl)heterocyclyl;
L2 is C1-C6 saturated alkylene or C2-C6 alkenylene, wherein the alkylene or alkenylene optionally may be substituted, provided that L2 is not xe2x80x94C(O)xe2x80x94, and wherein one of the carbon atoms of the alkylene optionally may be replaced by a heteroatom moiety selected from the group consisting of O; NRxe2x80x2, Rxe2x80x2 being alkyl, acyl, or hydrogen; S; S(O); or S(O)2;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted; and
Y2 is a chemical bond or a straight- or branched-chain saturated alkylene, which may be optionally substituted, provided that the alkylene is not substituted with a substituent of the formula xe2x80x94C(O)R wherein R comprises an xcex1-amino acyl moiety; and
Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation;
provided that when the carbon atom to which Cy is attached is oxo substituted, then Cy and Z are not both pyridyl.
Preferred substituents Cy, Ar, and Z according to this aspect of the invention are as defined above for the first embodiment. Preferred substituents Y2 are as defined above for Y1. In some preferred embodiments, L2 is saturated C1-C8 alkylene, more preferably C1-C6 alkylene, still more preferably C1-C4 alkylene, any of which groups may be optionally substituted. In some other preferred embodiments, L2 is C2-C8 alkenylene, more preferably C2-C6 alkenylene, and still more preferably C2-C8 alkenylene, any of which groups may be optionally substituted. The alkylene or alkenylene group may be substituted at one or more carbon positions with a substituent preferably selected from the list of preferred substituents recited above. More preferably, L2 is substituted at one or two positions with a substituent independently selected from the group consisting of C1-C6 alkyl, C6-C10 aryl, amino, oxo, hydroxy, C1-C4 alkoxy, and C6-C10 aryloxy. In some particularly preferred embodiments, the alkylene or alkenylene group is substituted with one or two oxo or hydroxy groups. However, L2 preferably is not xe2x80x94C(O)xe2x80x94, and when the carbon atom to which Cy is attached is oxo substituted, Cy and Z preferably are not both pyridyl.
In some preferred embodiments, L1 is C1-C6 saturated alkylene, wherein on of the carbon atoms of the saturated alkylene is replaced by a heteroatom moiety selected from the group consisting of O; NRxe2x80x2, Rxe2x80x2 being alkyl, acyl, or hydrogen; S; S(O); or S(O)2. Preferably, the carbon atom adjacent to Cy is replaced by a heteroatom moiety. In some particularly preferred embodiments, L1 is selected from the group consisting of xe2x80x94Sxe2x80x94(CH2)2xe2x80x94, xe2x80x94S(O)xe2x80x94(CH2)2xe2x80x94, xe2x80x94S(O)2xe2x80x94(CH2)2xe2x80x94, xe2x80x94Sxe2x80x94(CH2)3xe2x80x94, xe2x80x94S(O)xe2x80x94(CH2)3xe2x80x94, and xe2x80x94S(O)2xe2x80x94(CH2)3xe2x80x94.
In a third embodiment, the invention provides novel inhibitors of histone deacetylase represented by formula (3):
Cyxe2x80x94L3xe2x80x94Arxe2x80x94Y3xe2x80x94C(O)NHxe2x80x94Zxe2x80x83xe2x80x83(3)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted, provided that Cy is not a (spirocycloalkyl)heterocyclyl;
L3 is selected from the group consisting of
(a) xe2x80x94(CH2)mxe2x80x94Wxe2x80x94, where m is 0, 1, 2, 3, or 4, and W is selected from the group consisting of C(O)NHxe2x80x94, xe2x80x94S(O)2NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94NHS(O)2xe2x80x94, and xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94; and
(b) C1-C6 alkylene or C2-C6 alkenylene, wherein the alkylene or alkenylene optionally may be substituted, provided that L3 is not xe2x80x94C(O)xe2x80x94, and wherein one of the carbon atoms of the alkylene optionally may be replaced by O; NRxe2x80x2, Rxe2x80x2 being alkyl, acyl, or hydrogen; S; S(O); or S(O)2;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted; and
Y3 is C2 alkenylene or C2 alkynylene, wherein one or both carbon atoms of the alkenylene optionally may be substituted with alkyl, aryl, alkaryl, or aralkyl; and
Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation;
provided that when Cy is unsubstituted phenyl, Ar is not phenyl wherein L3 and Y3 are oriented ortho or meta to each other.
Preferred substituents Cy, Ar, and Z according to this aspect of the invention are as defined above for the first embodiment. Preferred substituents L3 are as defined above for L1 or L2.
Preferably, Y3 is C2 alkenylene or C2 alkynylene, wherein one or both carbon atoms of the alkenylene optionally may be substituted with C1-C6 alkyl, C6-C10 aryl, (C1-C6)alk(C6-C10)aryl, or (C6-C10)ar(C1-C6)alkyl. More preferably, Y3 is C2 alkenylene or C2 alkynylene, wherein one or both carbon atoms of the alkenylene optionally may be substituted with C1-C4 alkyl, C6-C14 aryl, (C1-C4)alk(C6-C10)aryl, or (C6-C10)arxe2x80x94(C1-C4)alkyl. Still more preferably, Y3 is selected from the group consisting of xe2x80x94Cxe2x89xa1Cxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94C(CH3)xe2x95x90CHxe2x80x94, and xe2x80x94CHxe2x95x90C(CH3)xe2x80x94.
Compounds of formula Cyxe2x80x94L1xe2x80x94Arxe2x80x94Y1xe2x80x94C(O)xe2x80x94NHxe2x80x94Oxe2x80x94M, wherein L1 is xe2x80x94S(O)2NHxe2x80x94, preferably may be prepared according to the synthetic routes depicted in Schemes 1-3. Accordingly, in certain preferred embodiments, compounds I are preferably prepared according to the general synthetic route depicted in Scheme 1. Thus, a sulfonyl chloride (II) is treated with an amine (III) in a solvent such as methylene chloride in the presence of an organic base such as triethylamine. Treatment of the crude product with a base such as sodium methoxide in an alcoholic solvent such as methanol effects cleavage of any dialkylated material and affords the sulfonamide (IV). Hydrolysis of the ester function in IV can be effected by treatment with a hydroxide base, such as lithium hydroxide, in a solvent mixture such as tetrahydrofuran and methanol to afford the corresponding acid (V). 
In some embodiments, conversion of the acid V to the hydroxamic acid I may be accomplished by coupling V with a protected hydroxylamine, such as tetrahydropyranylhydroxylamine (NH2OTHP), to afford the protected hydroxamate VI, followed by acidic hydrolysis of VI to provide the hydroxamic acid I. The coupling reaction is preferably accomplished with the coupling reagent dicyclohexylcarbodiimide (DCC) in a solvent such as methylene chloride (Method A) or with the coupling reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in presence of N-hydroxy benzotriazole in an aprotic solvent such as dimethylformamide (Method D). Other coupling reagents are known in the art and may also be used for this reaction. Hydrolysis of VI is preferably effected by treatment with an organic acid such as camphorsulfonic acid in a protic solvent such as methanol.
Alternatively, in some other embodiments, acid V is converted to the corresponding acid chloride, preferably by treatment with oxalic chloride, followed by the addition of a protected hydroxylamine such as O-trimethylsilyl-hydroxylamine in a solvent such as methylene chloride, which then provides the hydroxylamine I upon workup (Method C).
In still other embodiments, the ester IV is preferably treated with hydroxylamine in a solvent such as methanol in the presence of a base such as sodium methoxide to furnish the hydroxylamine I directly (Method B). 
Compounds of formula X and XIV preferably are prepared according to the general procedure outlined in Scheme 2. Thus, an aminoaryl halide (VII) is treated with a sulfonyl chloride in presence of a base such as triethylamine, followed by treatment with an alkoxide base, to furnish the sulfonamide VIII. One of skill in the art will recognize that reverse sulfonamide analogs can be readily prepared by an analogous procedure, treating a haloarenesulfonyl halide with an arylamine.
Compound VIII is coupled with a terminal acetylene or olefinic compound in the presence of a palladium catalyst such as tetrakis(triphenylphosphine)palladium(0) in a solvent such as pyrrolidine to afford IX.
Oxidation of the compound of formula IX (X=CH2OH), followed by homologation of the resulting aldehyde using a Wittig type reagent such as carbethoxymethylenetriphenylphosphorane in a solvent such as acetonitrile, gives the compound of formula XI. Basic hydrolysis of XI, such as by treatment with lithium hydroxide in a mixture of THF and water, provides the acid XII. Hydrogenation of XII may preferably be performed over a palladium catalyst such as Pd/C in a protic solvent such as methanol to afford the saturated acid XIII. Coupling of the acid XIII with an O-protected hydroxylamine such as O-tetrahydropyranylhydroxylamine is effected by treatment with a coupling reagent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in the presence of N-hydroxybenzotriazole (HOBT), or N,N-dicyclohexylcarbodiimide (DCC), in a solvent such as DMF, followed by deprotection to furnish the compound of general formula XIV.
The acid IX, wherein X=COOH, may be coupled directly with an O-protected hydroxylamine such as O-tetrahydropyranylhydroxylamine, followed by deprotection of the hydroxy protecting group to furnish the hydroxamic acid X.
Compounds of formula Cyxe2x80x94L1xe2x80x94Arxe2x80x94Y1xe2x80x94C(O)xe2x80x94NHxe2x80x94Oxe2x80x94M, wherein L1 is xe2x80x94C(O)NHxe2x80x94, preferably may be prepared according to the synthetic routes analogous to those depicted in Schemes 1-2, substituting acid chloride starting materials for the sulfonyl chloride starting materials in those Schemes. 
Compounds of the formula Cyxe2x88x92L2xe2x80x94Arxe2x80x94Y2xe2x80x94C(O)xe2x80x94NHxe2x80x94Oxe2x80x94M are preferably prepared according to the synthetic routes outlined in Schemes 3-5. Accordingly, in certain preferred embodiments, compounds of formulae XIX and XXI (L2=xe2x80x94C(O)xe2x80x94CHxe2x95x90CHxe2x80x94 or xe2x80x94C(O)CH2CHxe2x80x94) preferably are prepared according to the route described in Scheme 3. Thus, a substituted aryl acetophenone (XV) is treated with an aryl aldehyde (XVI) in a protic solvent such as methanol in the presence of a base such as sodium methoxide to afford the enone XVII.
The acid substituent of XVII (R=H) is coupled with an O-protected hydroxylamine such as O-tetrahydropyranylhydroxylamine (R1=tetrahydropyranyl) to afford the O-protected-N-hydroxybenzamide XVIII. The coupling reaction is preferably performed by treating the acid and hydroxylamine with dicyclohexylcarbodiimide in a solvent such as methylene chloride or with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in the presence of N-hydoxybenzotriazole in a solvent such as dimethylformamide. Other coupling reagents are known in the art and may also be used in this reaction. O-Deprotection is accomplished by treatment of XVIII with an acid such as camphorsulfonic acid in a solvent such as methanol to afford the hydroxamic acid XIX (L2=xe2x80x94C(O)xe2x80x94CHxe2x95x90CHxe2x80x94).
Saturated compounds of formula XXI (L2=xe2x80x94C(O)xe2x80x94CH2CH2xe2x80x94) are preferably prepared by hydrogenation of XVII (R=Me) over a palladium catalyst, such as 10% Pd/C, in a solvent such as methanol-tetrahydrofuran. Basic hydrolysis of the resultant product XIX with lithium hydroxide, followed by N-hydroxy amide formation and acid hydrolysis as described above, then affords the hydroxamic acid XXI. 
Compounds of formula XXVI (L2=CH2)o+2xe2x80x94) are preferably prepared by the general procedures described in Schemes 4 and 5. Thus, in some embodiments, a terminal olefin (XXII) is coupled with an aryl halide (XXIII) in the presence of a catalytic amount of a palladium source, such as palladium acetate or tris(dibenzylideneacetone)dipalladium(0), a phosphine, such as triphenylphosphine, and a base, such as triethylamine, in a solvent such as acetonitrile to afford the coupled product XXIV. Hydrogenation, followed by N-hydroxyamide formation and acid hydrolysis, as described above, affords the hydroxamic acid XXVI. 
Alternatively, in some other embodiments, a phosphonium salt of formula XXVII is treated with an aryl aldehyde of formula XXVIII in the presence of base, such as lithium hexamethyldisilazide, in a solvent, such as tetrahydrofuran, to produce the compound XXIV. Hydrogenation, followed by N-hydroxyamide formation and acidic hydrolysis, then affords the compounds XXVI. 
Compounds of formula Cyxe2x80x94Lxe2x80x94Arxe2x80x94Yxe2x80x94C(O)xe2x80x94NHxe2x80x94Z, wherein L is L1 or L2, Y is Y1 or Y2, and Z is anilinyl or pyridyl, are preferably prepared according to synthetic routes outlined in Scheme 6. An acid of formula Cyxe2x80x94Lxe2x80x94Arxe2x80x94Yxe2x80x94C(O)xe2x80x94OH (XXIX), prepared by one of the methods shown in Schemes 1-5, is converted to the corresponding acid chloride XXX according to standard methods, e.g., by treatment with sodium hydride and oxalyl chloride. Treatment of XXX with 2-aminopyridine and a tertiary base such as N-methylmorpholine, preferably in dichloromethane at reduced temperature, then affords the pyridyl amide XXXI. In a similar fashion, the acid chloride XXX may be treated with 1,2-phenylenediamine to afford the anilinyl amide XXXII. Alternatively, the acid chloride XXX may be treated with a mono-protected 1,2-phenylenediamine, such as 2-(t-BOC-amino)aniline, followed by deprotection, to afford XXXII.
In another alternative procedure, the acid XXIX may be activated by treatment with carbonyldiimidazole (CDI), followed by treatment with 1,2-phenylenediamine and trifluoroacetic acid to afford the anilinyl amide XXXII. 
Compounds of formula XXXVIII (L2=xe2x80x94C(O)-alkylene-) preferably are prepared according to the general procedure depicted in Scheme 7. Thus, Aldol condensation of ketone XXXIII (R1=H or alkyl) with aldehyde XXXIV affords the adduct XXXV. The adduct XXXV may be directly converted to the corresponding hydroxamic acid XXXVI, or may first undergo hydrogenation to afford the saturated compound XXVII and then be converted to the hydroxamic acid XXXVIII. 
Compounds of formula (2), wherein one of the carbon atoms in L2 is replaced with S, S(O), or S(O)2 preferably are prepared according to the general procedure outlined in Scheme 8. Thus, thiol XXXIX is added to olefin XL to produce XLI. The reaction is preferably conducted in the presence of a radical initiator such as 2,2xe2x80x2-azobisisobutyronitrile (AIBN) or 1,1xe2x80x2-azobis(cyclohexanecarbonitrile) (VAZO(trademark)). Sulfide oxidation, preferably by treatment with m-chloroperbenzoic acid (mCPBA), affords the corresponding sulfone, which is conveniently isolated after conversion to the methyl ester by treatment with diazomethane. Ester hydrolysis then affords the acid XLII, which is converted to the hydroxamic acid XLIII according to any of the procedures described above. The sulfide XLI also may be converted directly to the corresponding hydroxamic acid XLIV, which then may be selectively oxidized to the sulfoxide XLV, for example, by treatment with hydrogen peroxide and tellurium dioxide.
In a second aspect, the invention provides pharmaceutical compositions comprising an inhibitor of histone deacetylase represented by any one of formulae (1)-(6) and a pharmaceutically acceptable carrier, excipient, or diluent. Compounds of the invention may be formulated by any method well known in the art and may be prepared for administration by any route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, intratracheal, or intrarectal. In certain preferred embodiments, compounds of the invention are administered intravenously in a hospital setting. In certain other preferred embodiments, administration may preferably be by the oral route.
The characteristics of the carrier will depend on the route of administration. As used herein, the term xe2x80x9cpharmaceutically acceptablexe2x80x9d means a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism, and that does not interfere with the effectiveness of the biological activity of the active ingredient(s). Thus, compositions according to the invention may contain, in addition to the inhibitor, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The preparation of pharmaceutically acceptable formulations is described in, e.g., Remington""s Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990.
In a third aspect, the invention provides a method of inhibiting histone deacetylase in a cell, comprising contacting a cell in which inhibition of histone deacetylase is desired with an inhibitor of histone deacetylase according to the invention. In a first embodiment according to this aspect of the invention, the inhibitor of histone deacetylase is represented by the formula (1)
Cyxe2x80x94L1xe2x80x94Arxe2x80x94Y1xe2x80x94C(O)xe2x80x94NHxe2x80x94Zxe2x80x83xe2x80x83(1)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted;
L1 is xe2x80x94(CH2)nxe2x80x94Wxe2x80x94, where m is 0, 1, 2, 3, or 4, and W is selected from the group consisting of C(O)NHxe2x80x94, xe2x80x94S(O)2NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94NHS(O)2xe2x80x94, and xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted;
Y1 is a chemical bond or a straight- or branched-chain saturated alkylene, wherein said alkylene may be optionally substituted; and
Z is selected from the group consisting of anilinyl, pyridyl, 2-thioxo-1,3,4-thiadiazol-2-yl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation;
provided that when L1 is xe2x80x94C(O)NHxe2x80x94, Y is xe2x80x94(CH2)nxe2x80x94, n being 1, 2, or 3, and Z is xe2x80x94Oxe2x80x94M, then Cy is not aminophenyl, dimethylaminophenyl, or hydroxyphenyl.
In a second embodiment according to this aspect of the invention, the inhibitor of histone deacetylase is represented by formula (2):
Cyxe2x80x94L2xe2x80x94Arxe2x80x94Y2xe2x80x94C(O)NHxe2x80x94Zxe2x80x83xe2x80x83(2)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted;
L2 is C1-C6 saturated alkylene or C2-C6 alkenylene, either of which may be optionally substituted;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted; and
Y2 is a chemical bond or a straight- or branched-chain saturated alkylene, which may be optionally substituted, provided that the alkylene is not substituted with a substituent of the formula xe2x80x94C(O)R wherein R comprises an xcex1-amino acyl moiety; and
Z is selected from the group consisting of anilinyl, pyridyl, 2-thioxo-1,3,4-thiadiazol-2-yl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation.
In a third embodiment according to this aspect of the invention, the inhibitor of histone deacetylase is represented by the formula (3):
Cyxe2x80x94L3xe2x80x94Arxe2x80x94Y3xe2x80x94C(O)NHxe2x80x94Zxe2x80x83xe2x80x83(3)
wherein
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted, provided that Cy is not a (spirocycloalkyl)heterocyclyl;
L3 is selected from the group consisting of
(a) xe2x80x94(CH2)nxe2x80x94Wxe2x80x94, where m is 0, 1, 2, 3, or 4, and W is selected from the group consisting of xe2x80x94C(O)NHxe2x80x94, xe2x80x94S(O)2NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94NHS(O)2xe2x80x94, and xe2x80x94NHxe2x80x94C(O)xe2x80x94NHxe2x80x94; and
(b) C1-C6 alkylene or C2-C6 alkenylene, wherein the alkylene or alkenylene optionally may be substituted, provided that L3 is not xe2x80x94C(O)xe2x80x94, and wherein one of the carbon atoms of the alkylene optionally may be replaced by O; NRxe2x80x2, Rxe2x80x2 being alkyl, acyl, or hydrogen; S; S(O); or S(O)2;
Ar is arylene, wherein said arylene optionally may be additionally substituted and optionally may be fused to an aryl or heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be optionally substituted; and
Y3 is C2 alkenylene or C2 alkynylene, wherein one or both carbon atoms of the alkenylene optionally may be substituted with alkyl, aryl, alkaryl, or aralkyl; and
Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and xe2x80x94Oxe2x80x94M, M being H or a pharmaceutically acceptable cation;
provided that when Cy is unsubstituted phenyl, Ar is not phenyl wherein L3 and Y3 are oriented ortho or meta to each other.
In a fourth embodiment according to this aspect of the invention, the novel histone deacetylase inhibitor is selected from the group represented by formulae (4)-(6): 
Measurement of the enzymatic activity of a histone deacetylase can be achieved using known methodologies. For example, Yoshida et al., J. Biol. Chem., 265: 17174-17179 (1990), describes the assessment of histone deacetylase enzymatic activity by the detection of acetylated histones in trichostatin. A treated cells. Taunton et al., Science, 272: 408-411 (1996), similarly describes methods to measure histone deacetylase enzymatic activity using endogenous and recombinant HDAC-1. Both of these references are hereby incorporated by reference in their entirety.
In some preferred embodiments, the histone deacetylase inhibitor interacts with and reduces the activity of all histone deacetylases in the cell. In some other preferred embodiments according to this aspect of the invention, the histone deacetylase inhibitor interacts with and reduces the activity of fewer than all histone deacetylases in the cell. In certain preferred embodiments, the inhibitor interacts with and reduces the activity of one histone deacetylase (e.g., HDAC-1), but does not interact with or reduce the activities of other histone deacetylases (e.g., HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, and HDAC-8). As discussed below, certain particularly preferred histone deacetylase inhibitors are those that interact with and reduce the enzymatic activity of a histone deacetylase that is involved in tumorigenesis. Certain other preferred histone deacetylase inhibitors interact with and reduce the enzymatic activity of a fungal histone deacetylase.
Preferably, the method according to the third aspect of the invention causes an inhibition of cell proliferation of the contacted cells. The phrase xe2x80x9cinhibiting cell proliferationxe2x80x9d is used to denote an ability of an inhibitor of histone deacetylase to retard the growth of cells contacted with the inhibitor as compared to cells not contacted. An assessment of cell proliferation can be made by counting contacted and non-contacted cells using a Coulter Cell Counter (Coulter, Miami, Fla.) or a hemacytometer. Where the cells are in a solid growth (e.g., a solid tumor or organ), such an assessment of cell proliferation can be made by measuring the growth with calipers and comparing the size of the growth of contacted cells with non-contacted cells.
Preferably, growth of cells contacted with the inhibitor is retarded by at least 50% as compared to growth of non-contacted cells. More preferably, cell proliferation is inhibited by 100% (i.e., the contacted cells do not increase in number). Most preferably, the phrase xe2x80x9cinhibiting cell proliferationxe2x80x9d includes a reduction in the number or size of contacted cells, as compared to non-contacted cells. Thus, an inhibitor of histone deacetylase according to the invention that inhibits cell proliferation in a contacted cell may induce the contacted cell to undergo growth retardation, to undergo growth arrest, to undergo programmed cell death (i.e., to apoptose), or to undergo necrotic cell death.
The cell proliferation inhibiting ability of the histone deacetylase inhibitors according to the invention allows the synchronization of a population of asynchronously growing cells. For example, the histone deacetylase inhibitors of the invention may be used to arrest a population of non-neoplastic cells grown in vitro in the G1 or G2 phase of the cell cycle. Such synchronization allows, for example, the identification of gene and/or gene products expressed during the G1 or G2 phase of the cell cycle. Such a synchronization of cultured cells may also be useful for testing the efficacy of a new transfection protocol, where transfection efficiency varies and is dependent upon the particular cell cycle phase of the cell to be transfected. Use of the histone deacetylase inhibitors of the invention allows the synchronization of a population of cells, thereby aiding detection of enhanced transfection efficiency.
In some preferred embodiments, the contacted cell is a neoplastic cell. The term xe2x80x9cneoplastic cellxe2x80x9d is used to denote a cell that shows aberrant cell growth. Preferably, the aberrant cell growth of a neoplastic cell is increased cell growth. A neoplastic cell may be a hyperplastic cell, a cell that shows a lack of contact inhibition of growth in vitro, a benign tumor cell that is incapable of metastasis in vivo, or a cancer cell that is capable of metastasis in vivo and that may recur after attempted removal. The term xe2x80x9ctumorigenesisxe2x80x9d is used to denote the induction of cell proliferation that leads to the development of a neoplastic growth. In some embodiments, the histone deacetylase inhibitor induces cell differentiation in the contacted cell. Thus, a neoplastic cell, when contacted with an inhibitor of histone deacetylase may be induced to differentiate, resulting in the production of a daughter cell that is phylogenetically more advanced than the contacted cell.
In some preferred embodiments, the contacted cell is in an animal. Thus, the invention provides a method for treating a cell proliferative disease or condition in an animal, comprising administering to an animal in need of such treatment a therapeutically effective amount of a histone deacetylase inhibitor of the invention. Preferably, the animal is a mammal, more preferably a domesticated mammal. Most preferably, the animal is a human.
The term xe2x80x9ccell proliferative disease or conditionxe2x80x9d is meant to refer to any condition characterized by aberrant cell growth, preferably abnormally increased cellular proliferation. Examples of such cell proliferative diseases or conditions include, but are not limited to, cancer, restenosis, and psoriasis. In particularly preferred embodiments, the invention provides a method for inhibiting neoplastic cell proliferation in an animal comprising administering to an animal having at least one neoplastic cell present in its body a therapeutically effective amount of a histone deacetylase inhibitor of the invention.
It is contemplated that some compounds of the invention have inhibitory activity against a histone deacetylase from a protozoal source. Thus, the invention also provides a method for treating or preventing a protozoal disease or infection, comprising administering to an animal in need of such treatment a therapeutically effective amount of a histone deacetylase inhibitor of the invention. Preferably the animal is a mammal, more preferably a human. Preferably, the histone deacetylase inhibitor used according to this embodiment of the invention inhibits a protozoal histone deacetylase to a greater extent than it inhibits mammalian histone deacetylases, particularly human histone deacetylases.
The present invention further provides a method for treating a fungal disease or infection comprising administering to an animal in need of such treatment a therapeutically effective amount of a histone deacetylase inhibitor of the invention. Preferably the animal is a mammal, more preferably a human. Preferably, the histone deacetylase inhibitor used according to this embodiment of the invention inhibits a fungal histone deacetylase to a greater extent than it inhibits mammalian histone deacetylases, particularly human histone deacetylases.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d is meant to denote a dosage sufficient to cause inhibition of histone deacetylase activity in the cells of the subject, or a dosage sufficient to inhibit cell proliferation or to induce cell differentiation in the subject. Administration may be by any route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, intratracheal, or intrarectal. In certain particularly preferred embodiments, compounds of the invention are administered intravenously in a hospital setting. In certain other preferred embodiments, administration may preferably be by the oral route.
When administered systemically, the histone deacetylase inhibitor is preferably administered at a sufficient dosage to attain a blood level of the inhibitor from about 0.01 M to about 100 M, more preferably from about 0.05 M to about 50 M, still more preferably from about 0.1 M to about 25 M, and still yet more preferably from about 0.5 M to about 25 M. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. One of skill in the art will appreciate that the dosage of histone deacetylase inhibitor necessary to produce a therapeutic effect may vary considerably depending on the tissue, organ, or the particular animal or patient to be treated.
In certain preferred embodiments of the fifth and sixth aspects of the invention, the method further comprises contacting the cell with an antisense oligonucleotide that inhibits the expression of a histone deacetylase. The combined use of a nucleic acid level inhibitor (i.e., antisense oligonucleotide) and a protein level inhibitor (i.e., inhibitor of histone deacetylase enzyme activity) results in an improved inhibitory effect, thereby reducing the a mounts of the inhibitors required to obtain a given inhibitory effect as compared to the amounts necessary when either is used individually. The antisense oligonucleotides according to this aspect of the invention are complementary to regions of RNA or double-stranded DNA that encode HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC7, and/or HDAC-8.
For purposes of the invention, the term xe2x80x9coligonucleotidexe2x80x9d includes polymers of two or more deoxyribonucleosides, ribonucleosides, or 2xe2x80x2-O-substituted ribonucleoside residues, or any combination thereof. Preferably, such oligonucleotides have from about 6 to about 100 nucleoside residues, more preferably from about 8 to about 50 nucleoside residues, and most preferably from about 12 to about 30 nucleoside residues. The nucleoside residues may be coupled to each other by any of the numerous known internucleoside linkages. Such internucleoside linkages include without limitation phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate and sulfone internucleoside linkages. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof. The term oligonucleotide also encompasses such polymers having chemically modified bases or sugars and/or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines and adamantane. For purposes of the invention the term xe2x80x9c2xe2x80x2-O-substitutedxe2x80x9d means substitution of the 2xe2x80x2 position of the pentose moiety with an xe2x80x94O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an xe2x80x94O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or such 2xe2x80x2 substitution may be with a hydroxy group (to produce a ribonucleoside), an amino or a halo group, but not with a 2xe2x80x2-H group. The term xe2x80x9coligonucleotidexe2x80x9d also encompasses linked nucleic acid and peptide nucleic acid.
Particularly preferred antisense oligonucleotides utilized in this aspect of the invention include chimeric oligonucleotides and hybrid oligonucleotides.
For purposes of the invention, a xe2x80x9cchimeric oligonucleotidexe2x80x9d refers to an oligonucleotide having more than one type of internucleoside linkage. One preferred example of such a chimeric oligonucleotide is a chimeric oligonucleotide comprising a phosphorothioate, phosphodiester or phosphorodithioate region, preferably comprising from about 2 to about 12 nucleotides, and an alkylphosphonate or alkylphosphonothioate region (see e.g., Pederson et al. U.S. Pat. Nos. 5,635,377 and 5,366,878). Preferably, such chimeric oligonucleotides contain at least three consecutive internucleoside linkages selected from phosphodiester and phosphorothioate linkages, or combinations thereof. For purposes of the invention, a xe2x80x9chybrid oligonucleotidexe2x80x9d refers to an oligonucleotide having more than one type of nucleoside. One preferred example of such a hybrid oligonucleotide comprises a ribonucleotide or 2xe2x80x2-O-substituted ribonucleotide region, preferably comprising from about 2 to about 12 2xe2x80x2-O-substituted nucleotides, and a deoxyribonucleotide region. Preferably, such a hybrid oligonucleotide will contain at least three consecutive deoxyribonucleosides and will also contain ribonucleosides, 2xe2x80x2-O-substituted ribonucleosides, or combinations thereof (see e.g., Metelev and Agrawal, U.S. Pat. No. 5,652,355).
The exact nucleotide sequence and chemical structure of an antisense oligonucleotide utilized in the invention can be varied, so long as the oligonucleotide retains its ability to inhibit expression of the gene of interest. This is readily determined by testing whether the particular antisense oligonucleotide is active by quantitating the mRNA encoding a product of the gene, or in a Western blotting analysis assay for the product of the gene, or in an activity assay for an enzymatically active gene product, or in a soft agar growth assay, or in a reporter gene construct assay, or an in vivo tumor growth assay, all of which are described in detail in this specification or in Ramchandani et al. (1997) Proc. Natl. Acad. Sci. USA 94: 684-689.
Antisense oligonucleotides utilized in the invention may conveniently be synthesized on a suitable solid support using well known chemical approaches, including H-phosphonate chemistry, phosphoramidite chemistry, or a combination of H-phosphonate chemistry and phosphoramidite chemistry (i.e., H-phosphonate chemistry for some cycles and phosphoramidite chemistry for other cycles). Suitable solid supports include any of the standard solid supports used for solid phase oligonucleotide synthesis, such as controlled-pore glass (CPG) (see, e.g., Pon, R. T. (1993) Methods in Molec. Biol. 20: 465-496).
Particularly, preferred oligonucleotides have nucleotide sequences of from about 13 to about 35 nucleotides which include the nucleotide sequences shown in Tables 1-3. Yet additional particularly preferred oligonucleotides have nucleotide sequences of from about 15 to about 26 nucleotides of the nucleotide sequences shown in Tables 1-3.